Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Summary

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Abstract

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNFα) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Introduction

Macrophages are innate immune cells, which play multiple roles in the immune response to infection or injury. Macrophages are responsible for initiating an immune response to infection or tissue damage, stopping the inflammatory response, and promoting the healing response¹. Examples of the three best studied macrophage activation states are: 1) macrophages treated with interferon gamma (IFNγ) and bacterial lipopolysaccharide (LPS), designated M(IFNγ + LPS), which contribute to the inflammatory response; 2) macrophages stimulated with interleukin 4 (IL-4), M(IL-4), which are associated with the healing response; 3) macrophages stimulated with immune complexes (IC) and LPS, M(IC + LPS), which have the ability to turn off the inflammatory response^2,3. M(IC + LPS) are distinct from M(IL-4) wound healing macrophages, and do not express the enzyme arginase (Arg-1) or FIZZ1⁴. The best marker for these anti-inflammatory macrophages is their cytokine production⁵. Macrophages have multiple roles in maintaining health, but also contribute to inflammatory diseases and cancer³. Because of this, macrophages are a key therapeutic target for the treatment of a wide variety of diseases. It is important to investigate the effects of antibodies on their activation state to develop macrophage-based treatments for disease.

The focus of this paper is on the use of murine bone marrow derived macrophages (BMDMs) and peritoneal macrophages to test the effect of antibody drugs on inflammatory responses in vitro and in vivo. Recently, there have been multiple studies showing the effects of antibodies on macrophage activation^6,7,8. Macrophages co-activated with immune complexes, which are antibodies complexed with an antigen, and LPS, a normally inflammatory stimulus, produce very high levels of the anti-inflammatory cytokine, IL-10, and very low levels of the pro-inflammatory cytokine, interleukin 12 (IL-12)⁹. In addition, infliximab, a monoclonal antibody against TNFα, has been found to work, in part, by inducing anti-inflammatory macrophages through its fragment crystallizable (Fc) region⁷. We have reported that IVIg + LPS induce anti-inflammatory macrophage activation that is similar to M(IC + LPS), wherein co-stimulated macrophages produce large amounts of IL-10 and low amounts of the pro-inflammatory cytokine subunit Interleukin 12 or 23 p40 (IL-12/23p40), interleukin- 6 (IL-6), and TNF⁸. IVIg is a drug comprised of polyclonal antibodies, primarily IgG, which has been pooled from the blood of more than 1,000 donors¹⁰. It is used to treat a wide variety of immunological diseases, such as idiopathic thrombocytopenic purpura and chronic demyelinating polyneuropathy, but its mechanism of action is not completely understood¹¹. The effects of antibody based drugs on macrophage activation can be assessed using methods described herein.

The effects of specific biologics on macrophage activation can be tested in BMDMs and peritoneal macrophages. Using these macrophage sources permits assessment of primary cells. Preliminary testing of antibodies on cultured primary cells requires less time and monetary investment than other time consuming and expensive disease models. By injecting a drug into a healthy mouse in vivo, and isolating the cells and analyzing them ex vivo, one can determine if studies are warranted to assess whether treatment with biologics affects macrophage activation in disease models.

With few studies testing the effect of biologic therapies on macrophage activation in vitro and in vivo directly, our techniques provide an advantage over alternative techniques. Current techniques involve testing biologic drug effects on mixed cell populations in vitro, such as the effect of infliximab in a mixed lymphocyte reaction (MLR) or IVIg on human macrophages in peripheral blood mononuclear cells, where the effect cannot be attributed to a specific cell type^7,12. Using BMDMs and peritoneal macrophages is advantageous over using cell lines, such as RAW264.7 cells, which do not produce the pro-inflammatory cytokine, IL-12, in response to LPS^8,13. Testing the effect of an antibody-based drug on macrophage responses ex vivo has advantages because cytokine responses can be attributed directly to macrophages, rather than inferring macrophage responses by measuring serum cytokine levels¹⁴. BMDMs and peritoneal macrophages can be derived and isolated from genetically modified mice to determine the specific role of a protein in anti-inflammatory macrophage activation. For example, we have used Il10 deficient(-/-) BMDMs to demonstrate that IVIg-induced reduction of pro-inflammatory cytokine production is partially dependent on IL-10⁸. A drug's mechanism of action can be investigated using western blotting, where the role of specific proteins and signaling events can be determined. Quantitative polymerase chain reaction (qPCR) can be performed on BMDMs or peritoneal macrophages to show patterns of gene expression that result from antibody activation. Disease models in mice can provide information on the potential efficacy of antibody-based biological therapies in models for diseases like inflammatory bowel disease, rheumatoid arthritis, and cancer^15,16,17. However, the techniques described here will provide information on the mechanism of action of these biologics by determining whether they induce anti-inflammatory macrophage activity.

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of British Columbia.

1. Bone Marrow Macrophage Derivation and Activation with Antibodies

1. Perform euthanasia using CO₂ asphyxiation.
   1. Place mouse in induction chamber. Euthanize mouse with 5% isoflurane anesthesia, for 60 - 90 s, until immobile and breathing is deep and slow.
   2. Turn off isoflurane anesthesia and administer 6 - 8 L/min of CO₂ until mouse has stopped breathing. Leave mouse with CO₂ on for at least another 5 min. Verify that there is no longer a heartbeat or respiration. Perform a cervical dislocation to ensure death.
      NOTE: 8 - 12 weeks old mice provide the highest yield of macrophages.
2. Spray mouse legs with 70% ethanol, and pin them with arms and legs stretched out in the supine position over a foam board. With scissors and forceps to hold the skin, make a shallow cut (0.2 cm) to remove the skin and fur from the surface of each of the hind legs.
   NOTE: Perform this procedure and all tissue culture manipulations in a sterile hood.
3. Trim muscle to make tibias and femurs visible. Remove the tibias and femurs, by cutting the bone and muscles just below the hip joint and above the ankles. Trim as much muscle off as possible.
4. Spray bones with 70% ethanol and wait 1 min for it to evaporate, then place them in 6 well plate of bone flush medium (Iscove's modified Dulbecco's medium (IMDM), 10% fetal bovine serum (FBS)).
5. Trim the ends of the bones by cutting 0.1 cm of bone off the ankle and top of the femur so that the bone cavity is exposed. Ensure that the marrow is visible and a 26 gauge needle can be placed in the bone cavity.
6. Cut the bones and muscles to separate the tibias and femurs from the knee joints. Insert a 26 gauge needle attached to a 10 mL syringe into the cavity. Flush the bone marrow into a 50 mL conical tube with 5 mL of bone flush medium. Flush two tibias and two femurs, from one mouse, into each 50 mL tube.
7. Pipette up and down several times or vortex the marrow at a slow speed to gently disperse clumps. Strings of marrow should be broken up into small (less than 0.5 mm) flecks of marrow. Fill the conical tube to 50 mL with bone flush medium. Pipette contents of the conical tube into a 75 cm² tissue culture treated flask, and incubate at 37 °C, 5% CO₂ for 1 h.
   NOTE: Mature macrophages and mesenchymal cells will become adherent to the flask and be discarded, while the desired hematopoietic progenitors remain in suspension.
8. Pipette the medium with hematopoietic progenitors into a 50 mL conical tube. Centrifuge the tube at 300 x g at room temperature for 5 min. Discard the supernatant and resuspend the pellet in 5 mL of macrophage colony stimulating factor (MCSF) culture medium (IMDM, 10% FBS, 5 ng/mL MCSF, 100 U/mL penicillin/streptomycin, and 150 μM monothioglycerol (MTG)).
9. Count cells using a hemocytometer¹⁸. Add medium to resuspend cells to a concentration of 0.5 x 10⁶ cells/mL, 1.5 x 10⁷ cells/flask in 30 mL of medium, and pipette up and down gently. Pipette 30 mL of the suspension into a new 75 cm² tissue culture treated flask.
10. On day 4 and day 7 after initial culture in step 1.9, verify that the cells are adherent and slightly branched using an inverted phase contrast bright field microscope. Discard the medium containing the non-adherent cells. Wash the cells with IMDM one time and add 30 mL of MCSF culture medium.
11. When the cells are mature by day 10 (>95% positive for F4/80 and Mac-1), verify again that the cells are adherent and slightly.
12. To plate cells for stimulations, remove the culture medium from the flask and add 8 mL of enzyme free, EDTA-based cell dissociation buffer (Table of Materials). Incubate the cells for 5 min at 37 °C, 5% CO₂.
13. Scrape cells gently, with a small amount of pressure, using a sterile cell scraper. Check in the microscope that cells have detached from the flask surface. Pipette dissociated cells into a 50 mL conical tube. Rinse the flask 3 times with 5 mL of IMDM and pool the rinse solution with the cells that were harvested. Centrifuge cells at 300 x g at room temperature for 5 min.
14. Resuspend the cell pellet in 3 mL of MCSF culture medium per 50 mL conical tube. Count viable cells using a hemocytometer¹⁸. Resuspend cells at a concentration of 1 x 10⁶ cells/mL and plate 100 μL of cell suspension (1 x 10⁵ cells/well) per well of a 96-well tissue culture treated, flat bottom plate.
    NOTE: Bones from one mouse will generate enough cells for 100 wells. If desired, 1 mL of cells can be plated in a 6-well plate (tissue culture treated, flat bottom). A larger number of cells (1 x 10⁶ cells) may be useful for western blot analyses.
15. Once adherent, stimulate duplicate or triplicate wells each with 10 ng/mL of LPS in IMDM, 30 mg/mL of IVIg, or IVIg + LPS. Doses of LPS or IVIg can be titrated to optimize responses. Leave duplicate or triplicate wells as unstimulated controls. Incubate them for 24 h (37 °C, 5% CO₂).
    NOTE: Re-plated cells should adhere to tissue culture wells within 1 h.
16. Collect cell supernatants from each well into individual 1.7 mL microcentrifuge tubes and remove any particulate matter by spinning at 10,000 x g for 5 min.
17. Remove the cell supernatant and avoid disturbing the pellet. Place the clarified cell supernatant in a sterile microcentrifuge tube.
    NOTE: Cell supernatants can be assayed for cytokines, IL-10, and cytokine subunit, IL-12/23p40, by enzyme-linked immunosorbent assay (ELISA) immediately, or stored at -80 °C long term. Follow ELISA protocol from commercially available kit (Table of Materials). Other pro-inflammatory cytokines can be assayed, such as IL-6 and TNF, as they are also reduced by co-treatment with IVIg + LPS stimulation compared to stimulation with LPS alone. After stimulation, adherent cells can be prepared for techniques such as western blotting or quantitative polymerase chain reaction (Q-PCR) ^19,20.

2. Challenging Mice In Vivo with IVIg and Harvesting Peritoneal Macrophages

1. Weigh each mouse. Calculate the volume of IVIg (100 mg/mL stock concentration) needed to provide a final dose of 2.5 g/kg body weight for each mouse.
   NOTE: For example, for a 20 g mouse, 500 µL of IVIg is required.
2. Draw the required amount of IVIg into a sterile 1 mL syringe fitted with a sterile 26 gauge needle. For control mice that will not receive IVIg, administer an equivalent dose volume of sterile phosphate buffered saline (PBS), pH 7.4 instead of IVIg.
3. Scruff the mouse with one hand by grabbing its skin at the back of the neck with your thumb and index finger and holding its tail and hind legs with your remaining fingers. Tilt its body towards the ground.
4. Place the needle at a 30 - 40° angle relative to the mouse's abdomen, in the lower right quadrant, bevel up, and insert 1.5 cm of the needle. Inject the IVIg or PBS intraperitoneally. Wait 1 h.
5. Euthanize mice with 5% isoflurane anesthesia followed by 6 - 8 L/min CO₂ asphyxiation (see steps 1.1.1 - 1.1.2), or according to institution's ethical guidelines.
6. Pin the mouse to a foam board, with limbs spread out. Spray the parts with 70% ethanol.
   NOTE: Perform the procedure in a sterile hood.
7. Make a shallow cut in the skin (0.2 cm) with scissors along the midline of the mouse, to avoid cutting the peritoneal cavity. Pull the abdominal skin off the mouse's belly using forceps, so that the lining of the intact peritoneal wall is visible.
8. Fill a 5 mL syringe with 5 mL of sterile PBS (pH 7.4). Insert a 25 or 27 gauge needle at the top of the cavity from either the left or right side toward the center of the mouse with the bevel of the needle up.
   NOTE: Place the needle carefully in the mouse so that you do not inject PBS into the organs, but rather, into the peritoneal space.
9. Push the fluid into the peritoneal cavity. Pull the needle out of the cavity and massage the peritoneum for 10 s to dislodge any cells. Insert needle at the top of the cavity and avoid organs. Collect and place the lavage fluid recovered into a 15 mL conical tube on ice.
   NOTE: For every 5 mL of fluid injected, approximately 3.75 mL will be recovered.
10. Repeat injection and recovery (steps 2.8 - 2.9) 3 more times (total injection volume of 20 mL),to recover 15 mL of lavage fluid in total. Collect lavage from each mouse into a separate 15 mL conical tube.
    NOTE: After the final injection, it may be easier to extract the fluid from the far left and right sides of the mouse, where fluid has accumulated. The organs will cause less interference with the needle at this position.
11. Spin cells down at 300 x g for 5 min at 4 °C. Resuspend the cells in 500 μL of plating medium (IMDM, 10% FBS, and 100 U/mL penicillin/streptomycin) per mouse flushed. Count viable macrophages using a hemocytometer¹⁸.
    NOTE: The macrophages will be slightly larger than the other peritoneal cells. Typical yield is 5 x 10⁵ - 1 x 10⁶ macrophages/mouse using a wild type C57BL/6 mouse.
    Optional: If a large number of red blood cells are present in the cell pellet, perform a lysis step to remove them, using the 1x red blood cell lysis buffer according to manufacturer's instructions.
12. Resuspend cells in plating medium at 1 x 10⁶ macrophages/mL and plate 100 μL per well in a 96-well flat bottom tissue culture treated plate.
    NOTE: It may be necessary to pool cells from more than one mouse for an experiment if using triplicate wells or titrating stimulants. For example, if one mouse had 5 x 10⁵ macrophages, 500 μL of plating medium would be required. There would be enough cells for 5 wells, or 1 experiment in duplicate, with one LPS dose.
13. Incubate cells for 1 h at 37 °C, 5% CO₂ to allow macrophages to become adherent. The adherent cells are the peritoneal macrophages. Remove cell supernatants and non-adherent cells. Rinse the wells twice with 200 μL IMDM (pre-warmed to 37 °C), wait 10 s and slowly tilt the plate. Replace the plating medium. Incubate the cells at 37 °C, 5% CO₂ for 30 min prior to stimulation.
14. Stimulate the macrophages with 10 ng/mL of LPS in IMDM, or leave unstimulated, as a control. Incubate for 24 h, collect and clarify cell supernatants (see steps 1.16 - 1.17) for IL-10 cytokine analysis by ELISA. Follow ELISA protocol from commercially available kit (Table of Materials).
    NOTE: After stimulation, adherent cells can be prepared for techniques such as western blot or PCR.

3. Challenging Mice in Vivo with IVIg + LPS and Harvesting Peritoneal Macrophages

1. Weigh each mouse. Calculate the volume of IVIg (100 mg/mL stock concentration) needed to provide a final dose of 2.5 g/kg body weight. Calculate the amount of LPS (100 μg/mL stock concentration in IMDM) needed to provide a final dose of 0.2 μg/g body weight.
   NOTE: For example, for a 20 g mouse, 500 µL of IVIg stock solution and 40 μL of a 100 μg/mL solution of LPS is required.
2. Draw the required amount of IVIg and LPS into a sterile 1 mL syringe fitted with a sterile 26 gauge needle. For control mice that will not receive IVIg, replace an equivalent volume of IVIg with sterile PBS pH 7.4.
3. Scruff the mouse with one hand by grabbing its skin at the back of the neck with your thumb and index finger and holding its tail and hind legs with your remaining fingers. Tilt its body towards the ground.
4. Place the needle at a 30 - 40° angle relative to the mouse's abdomen, in the lower right quadrant, bevel up, and insert 1.5 cm. Inject the IVIg + LPS, or PBS + LPS, intraperitoneally as in step 2.4.
5. After 1h, harvest peritoneal macrophages by performing steps 2.5 - 2.10.
6. Spin cells down at 300 x g for 5 min at 4 °C. Remove 1 mL of recovered lavage fluid and use for IL-10 and IL-12/23p40 analyses by ELISA according to manufacturer's instructions or freeze at -80 °C for future analyses.
   NOTE: To ensure accurate comparison between treatments for lavage fluid cytokine analysis, perform 5 mL flushs of the peritoneal cavity 4 times for a final volume of 15 mL. Not all fluid will be recovered from each flush.
7. As in step 2.11, resuspend and count viable macrophages.
   NOTE: The macrophages will be slightly larger than the other peritoneal cells. Typical yield is 5 x 10⁵ - 1 x 10⁶ macrophages/mouse using a wild type C57BL/6 mouse.
   Optional: If a large number of red blood cells are present in the cell pellet, perform a lysis step according to manufacturer's instructions to remove them, as in step 2.11.
8. As in step 2.12, re-suspend cells in plating medium.
   NOTE: It may be necessary to pool cells from more than one mouse for an experiment. For example, if one mouse had 5 x 10⁵ macrophages, 500 μL of plating medium would be required.
9. As in 2.13, incubate cells for 1 h at 37 °C 5% CO₂ to allow macrophages to become adherent.
10. Collect and clarify conditioned medium, as per steps 1.14 - 1.15, from all peritoneal cells.
    NOTE: Samples may be used immediately or frozen and stored at -80 °C for cytokine analysis by ELISA.
11. Perform step 2.11, but incubate cells for 24 h unstimulated. Collect and clarify cell supernatants, as per steps 1.16 - 1.17, for cytokine analysis by ELISA.
    NOTE: Cells can be prepared for techniques such as western blot or PCR, as in steps 1.16 - 1.17 and 2.14.

Results

Murine bone marrow derived macrophages can be cultured from hematopoietic cell precursors in bone marrow aspirates. Bone marrow aspirates pooled from femurs and tibias of one C57BL/6 mouse typically yield 10⁷ bone marrow derived macrophages, making them a convenient source of macrophages for experiments. BMDMs can be used to test antibody based drug responses when challenged with an inflammatory stimulus in vitro. Figure 1 shows that IVIg ...

Discussion

Macrophage activation states play an important role in both tissue homeostasis and disease²². Macrophages can have distinct as well as overlapping activation states, depending on cues in their microenvironment³. They have distinct roles in all stages of the inflammatory response: defense against pathogens, wound healing and tissue restitution, and also have a distinct anti-inflammatory activation state that is important for turning off the inflammatory response

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Iscove’s modified Dulbecco’s medium (IMDM)	Life technologies	12440053
Fetal Bovine Serum (FBS)	Life technologies	12483-020
Recombinant murine macrophage colony stimulating factor (MCSF)	Stemcell technologies	78059
Penicillin-streptomycin	Life technologies	15140148
Monothioglycerol (MTG)	Sigma	88640
1X red blood cell lysis buffer	eBioscience
Cell dissociation buffer	Life technologies	13150016	Enzyme-Free, Hanks's-based, EDTA
Lipopolysaccharide (LPS)	Sigma aldrich	L 4516	From E. coli 0127:B8
IVIg (Gammunex)	Grifols	Received from BC Children's Hospital, Transfusion Medicine
IVIg (Gammagard liquid)	Baxter Healthcare Corporation	Received from BC Children's Hospital, Transfusion Medicine
IVIg (Octagam)	Octapharma	Received from BC Children's Hospital, Transfusion Medicine
Phosphate buffered saline (PBS) (sterile), pH 7.4	Life technologies	10010023
mouse IL-10 ELISA	BD biosciences	555252
mouse IL-12/23p40 ELISA	BD biosciences	555165
anti-Erk1/2 antibody	Cell signalling technology	9101
anti-pp38 antibody	Cell signalling technology	9211
anti-GAPDH antibody	Fitzgerald industries	10R-G109A
26 g needle	BD biosciences	305110
1 mL syringe	BD biosciences	309659
10 mL syringe	BD biosciences	309604
15 mL conical tube	BD biosciences	352096
50 mL conical tube	BD biosciences	352070
microcentrifuge tube (1.7 mL)	Diamed	SPE155-N
75 cm2 tissue culture treated flask	BD biosciences	353136
Cell scraper	BD biosciences	353085
Forcepts	VWR	82027-386	fine tip, dissecting
Scissors	VWR	82027-582	Delicate, 4 1/2"
Brightfield microscope	Motic	AE31	Inverted phase contrast
Scale	Mettler	PE 3000